1. Field of the Invention
The present invention relates to a process for regenerating corn from mature embryos of many varieties of corn.
2. Description of the Prior Art
Plant regeneration from cells in culture is essential for the application of somatic hybridization, for the production of new varieties through somaclonal variation and for the use of genetic engineering in producing new varieties. Although plants can be regenerated from tissue culture of several varieties of corn, there are many varieties for which this has not been accomplished using similar techniques.
In recent years, plant cell culture successes have had a considerable influence on the understanding of the respective roles of cell and organism in control of plant growth and development. Isolated plant cells have been shown to be amenable to in vitro culture and complete plants have been regenerated from cultures derived from somatic tissues, either directly via somatic embryogenesis or indirectly via organogenesis. Generally the regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, especially growth regulators. Early investigations of certain plant species have suggested that exogenous auxin concentration is a major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of embryoid formation. In other species, exposure to a definite balance of auxin and cytokinin leads to the occurrence of organogenesis (shoots, then roots).
The process which has become the standard system for corn tissue culture is described by Green et al., Crop Science 15, 417 (1975). In this process, immature embryos were plated onto a callus induction medium which comprises the MS mineral salts, Straus vitamins and amino acids (glycine, asparagine, niacin, thiamine, pyridoxine and pantothenic acid), 2% sucrose, 0.8% agar and a hormone selected from 2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid (PCA), alpha-naphthaleneacetic acid (NAA), 2-isopentyladenine (2-ip) or mixtures thereof. Plantlets were regenerated by subculturing the callus on medium containing reduced hormone concentrations. Hormone concentrations which were useful were 3 mg/l 2,4-D and a mixture of 1 mg/l 2,4-D, 4 mg/l NAA and 0.05 mg/l 2-ip. Regeneration was then accomplished on medium containing 0.25 mg/l 2,4-D or a mixture of 1 mg/l NAA and 0.05 mg/l 2-ip, respectively. All culturing was conducted in a 16 hour light/8 hour dark cycle for 3-4 week intervals before transfer. This reference reports that callus induction did not occur in one of five genotypes tested.
Similar results with different media have been demonstrated by Freeling et al., Maydica 21, 97 (1976); Vasil et al., Theor. Appl.Genet. 66, 285 (1983); Edallo et al., Maydica 26, 39 (1981); Lu et al., Theor.Appl.Genet. 62, 109 (1982); Hibberd et al., Proc.Nat.Acad.Sci.USA 74, 5113 (1977); and Green et al., Crop Science 14, 54 (1974). The latter reference also demonstrates genotype effects on callus induction.
Although this procedure has been unsuccessful for regenerating plants from all maize genotypes, the regeneration of most genotypes is now possible through the substitution of dicamba for 2,4-D in the media. See published European Patent Application No. 0 177 738 and Duncan et al., Planta 165, 322 (1985).
Spontaneous variation has been observed among the regenerates. The variation is often termed somoclonal variation. Plant geneticists have been able to extract corn regenerates with agronomically desirable traits from the somoclonal varieties. Cowley et al., Agro.Abst. 1984:60; Gracen et al., Agro.Abst. 1984:68; and Lee et al., Agro.Abst. 1984:76. These regenerates have been used in corn breeding programs. In each instance, the regenerates were all obtained from embryogenic and organogenic calli derived from immature embryos, generally 10-15 days post-pollination.
Embryogenic and organogenic calli can be induced from a variety of corn plant tissues, in addition to immature embryos as described above. These plant tissues include anthers (Ting et al., Plant Sci.Lett. 23, 139 (1981)), glumes (Suprasenna et al., Theor.Appl.Genet. 72, 120 (1986)), leaf bases (Chang, Plant Cell Rep. 2, 183 (1983)), mesocotyls (Harms et al., Z.Pflangenzuchtg 77, 347 (1976)), seedling segments (Santos et al., Plant Sci. Letts. 33, 309 (1984)), immature ears (Molnar et al., Maize Genet. Corp.News Lett., 54, 52 (1980)), and immature tassels (Rhodes et al., Maize Genet.Corp.News Lett. 56, 148 (1982)). Regeneration of plants from these corn tissues, except immature embryos, has been inefficient. Immature embryo callus is very efficient for regeneration. All of these explant tissues are obtained from living plants, which is a disadvantage in that time and space are necessary to grow corn plants to obtain the desired tissue.
This disadvantage is overcome by the present invention, which utilizes the mature corn embryo as the explant tissue for the production of embryogenic and organogenic calli. Corn plants are regenerated from this calli. Use of dried mature embryos for corn regeneration is superior to other explant tissues since (a) it is not necessary to grow plants, and (b) callus can be induced from the mature embryo in about a week.